Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K d = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC 50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC 50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC 50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

The Ste20 family of serine/threonine kinases plays an important role in numerous cellular functions such as growth, apoptosis, and morphogenesis. We have identified a previously cloned but uncharacterized family member termed Stk10, which is a human homolog of murine Lok, a serine/threonine kinase highly expressed in lymphocytes. Northern analysis demonstrated that the Stk10 transcript is present in many tissues, although highest expression levels are seen in hematopoietic cells. Due to close sequence homology to human Slk and Xenopus laevis xPlkk1, two polo-like kinase kinases, we investigated whether Stk10 might also play a role as a Plk1 activator. Plk1 has been shown to be overexpressed in multiple tumor types, thus attracting high interest to its potential upstream regulators. We show here that Stk10 can associate with Plk1 in cells and furthermore can phosphorylate Plk1 in vitro. Engineered NIH-3T3 cell lines that overexpress a dominant negative version of Stk10 display an altered cell cycle phenotype characterized by increased DNA content, raising the possibility that expression of a dominant negative Stk10 may impinge upon Plk1 function in vivo; it has previously been shown that unregulated expression of Plk1 can result in a variety of nuclear defects. We suggest, therefore, that Stk10 is a novel polo-like kinase kinase that cooperates with hSlk to regulate Plk1 function in human cells.